title: Ultrahigh-resolution imaging reveals formation of neuronal SNARE/Munc18 complexes in situ.
abstract: Membrane fusion is mediated by complexes formed by SNAP-receptor (SNARE) and Secretory 1 (Sec1)/mammalian uncoordinated-18 (Munc18)-like (SM) proteins, but it is unclear when and how these complexes assemble. Here we describe an improved two-color fluorescence nanoscopy technique that can achieve effective resolutions of up to 7.5-nm full width at half maximum (3.2-nm localization precision), limited only by stochastic photon emission from single molecules. We use this technique to dissect the spatial relationships between the neuronal SM protein Munc18-1 and SNARE proteins syntaxin-1 and SNAP-25 (25 kDa synaptosome-associated protein). Strikingly, we observed nanoscale clusters consisting of syntaxin-1 and SNAP-25 that contained associated Munc18-1. Rescue experiments with syntaxin-1 mutants revealed that Munc18-1 recruitment to the plasma membrane depends on the Munc18-1 binding to the N-terminal peptide of syntaxin-1. Our results suggest that in a primary neuron, SNARE/SM protein complexes containing syntaxin-1, SNAP-25, and Munc18-1 are preassembled in microdomains on the presynaptic plasma membrane. Our superresolution imaging method provides a framework for investigating interactions between the synaptic vesicle fusion machinery and other subcellular systems in situ.
authors.affiliation: Structural Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA. PertsinA@mskcc.org
authors.forename: Alexandros
authors.initials: A
authors.lastname: Pertsinidis
authors.forename: Konark
authors.initials: K
authors.lastname: Mukherjee
authors.forename: Manu
authors.initials: M
authors.lastname: Sharma
authors.forename: Zhiping P
authors.initials: ZP
authors.lastname: Pang
authors.forename: Sang Ryul
authors.initials: SR
authors.lastname: Park
authors.forename: Yunxiang
authors.initials: Y
authors.lastname: Zhang
authors.forename: Axel T
authors.initials: AT
authors.lastname: Brunger
authors.forename: Thomas C
authors.initials: TC
authors.lastname: Südhof
authors.forename: Steven
authors.initials: S
authors.lastname: Chu
mesh_terms: D008856:Microscopy, Fluorescence / Q000379:methods*; D051938:Munc18 Proteins / Q000737:chemistry*; D050600:SNARE Proteins / Q000737:chemistry*; D050825:Synaptosomal-Associated Protein 25 / Q000737:chemistry; D050827:Syntaxin 1 / Q000737:chemistry
chemical_list: D051938:Munc18 Proteins; D050600:SNARE Proteins; D050825:Synaptosomal-Associated Protein 25; D050827:Syntaxin 1
keywords: active stabilization; colocalization; exocytosis; neurotransmission; single-molecule